Mitochondrial calcium (Ca2+) uptake is critical for the regulation of energy metabolism and mitochondrial movement, as well as buffering of intracellular Ca2+. This Ca2+ uptake is mediated by a highly selective Ca2+ pore complex consisting of mitochondrial calcium uniporter (MCU), the inner mitochondrial membrane protein, and its regulatory proteins. Although the mechanism how MCU and its regulatory components regulate mitochondrial Ca2+ uptake is well established, a mechanism underlying the regulation of mitochondrial movement by MCU still remains unrevealed.
Our previous work revealed that MCU-mediated mitochondrial Ca2+ content is a critical factor for the regulation of mitochondrial motility in the axon. Here, we find that MCU interacts with mitochondrial Rho GTPase 1 (Miro1), known to regulate mitochondrial motility. We identify the N-terminal domain of MCU, previously known as the mitochondrial targeting sequence (MTS), is essential for the interaction with Miro1.
Our results show that the N-terminus of MCU contains a potential transmembrane domain that allows interaction with Miro1, but that this is not required for the localization of MCU to mitochondria. Furthermore, our data show that elevation of intracellular Ca2+ causes the cleavage of the N-terminal domain of MCU, altering its interaction with Miro1, and this Ca2+-dependent MCU-Miro1 interaction is necessary to facilitate axonal mitochondrial mobility. Together, our findings reveal a novel functional relationship between Miro1 and MCU as a novel component of the MCU complex and a novel regulator of mitochondrial movement, respectively.
Introduction
Ca2+ uptake into the mitochondrial matrix is tightly regulated by the MCU complex, a Ca2+-selective channel, across the inner mitochondrial membrane (IMM)21,22. MCUb forms a hetero-oligomer with MCU, resulting in a decrease in Ca2+ uptake into the mitochondrial matrix in response to the cytosolic Ca2+ increase25. MICU1, as an activator or inhibitor of Ca2+ uptake via the MCU complex, is found in the IMS26.
MICU2, as the MCU gatekeeper, functions to inhibit the Ca2+ uptake into mitochondrial matrix at low cytosolic Ca2+28. It acts as a sensor of mitochondrial matrix Ca2+, allowing the MCU complex to inhibit Ca2+ uptake under normal condition29. EMRE interacts with MICU1 at the IMS, suggesting that EMRE cooperates with MICU1/MICU2 to regulate the MCU activity in response to Ca2+ on both sides of the IMM30.
The majority of Miro1 involving GTPase domains and EF hand domains are exposed on the cytoplasmic side of OMM34. In particular, Miro1 is critical in a long-distance axon and dendrites of the neuron, which. MCU and MCUb as pore-forming subunits form a highly selective Ca2+ channel with the regulatory subunits involving mitochondrial Ca2+ uptake protein 1, 2 (MICU1, MICU2), essential mitochondrial Ca2+ uniporter regulator (EMRE) and MCU regulator 1 (MCUR1).
The Miro1/TRAK complex interacts sequentially with kinesin or dynein motor proteins capable of anterograde or retrograde mitochondrial movement, respectively36. Although the molecular identity and functions of these machinery components are well established, a detailed mechanism underlying Ca 2+ -dependent regulation of the motor/adapter complex still remains unclear. So far, two models of Miro1 have been proposed to regulate mitochondrial mobility37.
The second model describes that the conformational change of Miro1 by Ca2+ binding to its EF hands allows mitochondria to dissociate from kinesin, while TRAK1/2 remains tethered to Miro1 with mitochondria37 (Figure 2B [ⅱ]). Miro1 is composed of two GTPase domains (GTPase1, GTPase2), two Ca 2+ -binding EF-hand domains (EF1, EF2) flanked by GTPase domains, and a single transmembrane (TM) domain at the C-terminus of Miro1. B) Schematic models of Miro1 for regulation of mitochondrial transport. ⅰ] Ca2+ binding to Miro1's EC hands triggers the dissociation of the motor protein, kinesin, from mitochondria, leading to detachment of the motor from microtubule channels.
Materials and Methods
Lipofectamine 2000/DNA complexes were incubated for 20 minutes at room temperature and added to cell culture plates. The pellet was removed and the supernatant containing intact mitochondria was centrifuged for 10 min at 10,000 x g at 4°C to obtain a purer mitochondrial fraction. For Proteinase K experiments, the mitochondrial pellet was incubated with Proteinase K (Roche diluted as 1:2000 in the isolation buffer for 10 min on ice.
Isolated mitochondria placed on prepared coverslips were incubated for 20 minutes to adhere the mitochondria to the coverslips, followed by fixation using 1× PBS containing 4%. Fixed mitochondria were permeabilized with 1× PBS containing 0.3% triton-X for 10 min at room temperature and blocked in 1× PBS containing 1% BSA and 0.05% sodium azide for 1 hour at room temperature. The next day, the coverslips were washed three times with PBST (0.1% TWEEN 20) for 10 minutes at room temperature.
Secondary Alexa antibodies diluted as 1:400 in the blocking solution were incubated for 2 h at room temperature with coverslip. For an experiment to examine MCU processing by Calcium, 8 hours after transfection, HEK293 cells were incubated with 2µM ionomycin for 10 minutes, and immediately fixed with 4% paraformaldehyde. After performing the Bradford assay (BioRad) for measuring protein concentration, 1 mg of proteins was incubated with 0.3 µl anti-Flag antibody (Sigma, F7425) or 1 µl anti-Myc antibody (Santa Cruz, 9E10) on rotator at 4° C overnight.
The immunoprecipitated protein-bead complex was then washed with 1 ml of the RIPA buffer and centrifuged for 5 min at 5,000 x g and 4°C. After repeating the wash five times, the immunoprecipitated proteins are eluted with SDS loading dye by heating at 95°C for 15 minutes. For Western blotting, SDS-PAGE gel consisting of 4% stacking gel and 12% running gel was used to separate proteins.
The next day, the membrane was washed three times with 1×TBST for 10 min at room temperature, followed by incubation with the secondary antibody diluted 1:2500 in 5% skim milk in 1×TBST for 1 h 30 min at . room temperature. For live imaging, a heating instrument was used to maintain 37°C, and the Zeiss Definite Focus z-correction device was used for axial stability. N2A cells were transfected with the vectors of interest, R-GECO and Mito-GEM-GECO, and incubated for at least 48 h.
Results
MCU interacts with Miro1 through MCU’s N-terminal domain
Although co-immunoprecipitation assay is an appropriate method to verify physiologically relevant MCU-Miro1 interaction, this assay, like in vitro biochemical methods, has difficulty identifying their interaction in intact mitochondria. FRET efficiency of Flag-MCU and Myc-Miro1 was significantly higher than that of either Flag-MCU and TOMM20-Myc or Flag-MCU(∆2-57a.a.) and Myc-Miro1, whereas Flag-MCU(mut1) with the mutation on DIME motif and Myc-Miro1 had similar level of FRET efficiency compared to that of Flag-MCU and Myc-Miro1 (Figure 3G). These results are consistent with co-immunoprecipitation assay analysis showing that Miro1 interacts with MCU through its N-terminal domain, not DIME motif.
Taken together, these data indicate that MCU and Miro1 exist in a single functional complex in mitochondria, and the N-terminal domain of MCU is essential for the interaction between MCU and Miro1. Protein extracts from MCU-Flag and Myc-Miro1 co-transfected HEK293 cells were immunoprecipitated with anti-Flag or Myc antibody conjugated to agarose A/G beads. Proteins produced from MCU(∆2-57a.a.)-Flag expression vector were detected only on the processed 35kDa band of MCU.
The interaction between MCU and Miro was not affected by mutations in the DIME motif (Mutant 1,2). F) Co-immunoprecipitation of MCU-Flag and Myc-Miro1 mutants. The Miro1 mutant having a deletion of the TM domain (∆TM) did not interact with the MCU, whereas the Miro1 mutant lacking 3a.a (∆KQR) still bound to the MCU. G) FRET analysis of Flag-MCU and Myc-Miro1 in extracted mitochondria. The FRET efficiency of Flag-MCU + Myc-Miro1 was significantly higher than that of Flag-MCU + TOMM20-Myc.
N-terminus of MCU has a transmembrane domain, which doesn’t have a critical role in mitochondrial targeting signal
Proteinase K efficiently digested not only TOMM20, the outer mitochondrial membrane protein, but also the Flag tag at the N-terminus of MCU. While intact mitochondria protected Mito-GFP, located in the mitochondrial matrix, and cytochrome C, located in the intermembrane space, from digestion by proteinase K (Figure 4E), confirming that the N-terminus of MCU can extend from the OMM and is located on the cytoplasmic side of this membrane. These results are consistent with the analysis using TMBASE which showed a predicted hydrophobic pathway (1-13a.a.) at the end of the N-terminus of MCU with the potential transmembrane domain (18-38a.a.) (Figure 4A).
Together, these data revealed that the N-terminal domain of MCU is not required for its mitochondrial localization and has a transmembrane domain, allowing it to interact with Miro1. The fluorescent signal of Flag-MCU, and not MCU-Flag, is strongly colocalized with that of Myc-Miro1. The fluorescent signal of Flag-MCU and TOMM20 disappeared by treatment with proteinase K, while that of Cytochrome C and Mito-GFP was still visible.
N= 96 mitochondria for TOMM20 without proteinase K, 51 for TOMM20 with proteinase K, 43 for Cytochrome C without proteinase K, 82 for Cytochrome C with proteinase K.
Intracellular Ca 2+ elevation results in the cleavage of MCU’s N-terminal domain
The ratio of raw MCU (Upper Band) to processed MCU (Lower Band) is shown as a Tukey's box profile. The N-terminal domain of MCU has the third transmembrane domain that allows it to localize toward the cytoplasmic side of the mitochondrial outer membrane. Under normal conditions, cleavage of the N-terminus of MCU does not occur, allowing it to retain the interaction with the transmembrane domain of Miro1.
However, intracellular Ca 2+ increase triggers the cleavage of N-terminal domain of MCU, resulting in loss of MCU-Miro1 interaction.
MCU-Miro1 interaction is essential for mitochondrial mobility in axons
The reduced speed caused by MCU knock-down was restored by expression of MCU-Flag, while MCU(∆2-57a.a.)-Flag cannot rescue the reduced mitochondrial speed in axons (Figure 6F,G). Taken together, these results imply that MCU-Miro1 interaction is essential for maintaining axonal mitochondrial mobility; however, cleaving the MCU's N-terminus and eliminating this interaction results in an increase in stationary mitochondria in axons. N2A cells were transfected with R-GECO and Mito-GEM-GECO as cytoplasmic and mitochondrial Ca 2+ indicator, respectively.
Quantification of the Mito-GEM-GECP ratio at the first frame after exposure of N2A cells to 2μM ionomycin. -dendra-labeled mitochondria in the cell body were photoconverted to red color to allow tracking of mitochondria moving from the cell body toward the axon.
Discussion
Bidirectional Ca2+dependent control of mitochondrial dynamics by Miro GTPase.
Acknowledgement
